Genome-wide Study of SBT Genes in Eight Plant Species: Evolution and Expression Outlines for Development and Stress Sulin Wen Shanghai Jiao Tong University https://orcid.org/0000-0002-5994-3439 Guanqun Chen Shanghai Jiao Tong University Tingting Huang Shanghai Jiao Tong University Xiaohui Shen ( [email protected] ) School of Design, Shanghai Jiao Tong University, Shanghai 200240, China. Research article Keywords: Subtilisin-like proteases, Genome-wide study, Evolution, Expression pattern, Arabidopsis, Rice Posted Date: October 8th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-77460/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/22 Abstract Background: Subtilisin-like proteases (or subtilases, SBT), a specic family of serine peptidases, are a very big family in plants. Previous studies showed association of SBTs with environment response and development. However, it is still unclear about the function and features of SBTs. Also, there is little information about SBTs classication or protein function research. Results: The characters of SBTs in eight plant species including Arabidopsis, rice, wheat etc. were analyzed. The analysis mainly contained evolutionary relationship, gene structure, chromosomal location, gene synteny and expression pattern. In short, the evolutionary trend of SBTs in the eight plant species was revealed. The expression pattern showed a scattered distribution of SBTs in diverse plant organs. Conclusions: Our results reveal classication, feature and expression pattern of SBT. Then the results might offer clues for future studies about SBTs and additionally related to their possible function in plants against stress and development. Background Protease is a kind of important enzyme in most living things, which breakdown of proteins into smaller polypeptides or single amino acids. Proteases are involved in many biological functions, including digestion of ingested proteins, protein catabolism, and cell signaling (King et al 2014, Shen et al 2006). Subtilisin-like protease (SBT), which was rst observed from Bacillus subtilis in 1947 (Lindstrom-Lang & Ottesen 1947), had been found in archaea, bacteria, eukaryote, fungi and yeast with its features like sequence, classication, function for the past decades (Coffeen & Wolpert 2004, Rawlings & Barrett 2000, Yamagata et al 1994), it was indicated an important protease in plants as well (Siezen & Leunissen 1997) . The rst plant SBT gene family was discovered in Lycopersicon esculentum (tomato) and was thought of 15 members (Meichtry et al 1999). Since then, in Arabidopsis thaliana, 56 known genes of this family was found (Rautengarten et al 2005), and 63 genes of this family in Oryza sativa (rice) was also indicated (Tripathi & Sowdhamini 2006). Besides, in the years that followed, the SBT family genes were explored in Physcomitrella patens (23 gene members), Populus trichocarpa (90 gene members), Vitis vinifera (82 gene members) and Solanum tuberosum (82 gene members), respectively (Figueiredo et al 2016, Schaller et al 2012, Sigrid Norero et al 2016). The function of SBT showed widely spread in plants. It was reported involved in all aspects of plant life cycle, such as development of seeds and fruits, cell wall modication, processing of peptide growth factors and epidermal development and make response to biotic and abiotic stress (Schaller et al 2012). With abiotic stimulus, SBT showed be involved in stress like drought and salt resistance mechanisms (Budic et al 2013, Che et al 2010, Liu & Howell 2010a, Liu & Howell 2010b, Liu et al 2007). A representative example is that two SBTs in common bean (Phaseolus vulgaris), PvSLP1 and PvSLP2, were responsible to drought (Budic et al 2013). Page 2/22 The SBT is constructed with three main domains, ie. Peptidase S8 domain(S8), protease-associated (PA) domain and inhibitor I9 domain (Figueiredo et al 2018). The peptidase S8 domain, also known as catalytic domain of SBT, is present in all SBTs with a highly conserved catalytic triad composed by aspartate (Asp), histidine (His) and serine (Ser) residues (Dodson & Wlodawer 1998). The PA domain is another main representative domain for SBT, consisting of an 120 to 160 amino acids insertion between the His and Ser active site residues (Luo & Hofmann 2001). Additionally, for the I9 domain(I9), its main function is keeping the inactive state of the zymogen and preventing the access of the substrate to the active site (Bryan 2002). Interaction between the PA and the I9 domains result in the cleavage of the N- terminus and it will open a access of substrates to the catalytic site, promoting the protease activity as well (Bergeron et al 2000). The SBTs were widespread in plants (Figueiredo et al 2018). Although some researches have revealed gene members, features and function in some plant species like tomato, rice, potato (Rautengarten et al 2005, Sigrid Norero et al 2016, Tripathi & Sowdhamini 2006), there are still many questions about this enzyme. For discovering SBTs in more detail, we performed bioinformatics tools to process a more complete analysis for SBT family in eight plant species, including Arabidopsis thaliana and 6 monocotsie. three main crops (Oryza sativa, Zea mays and Triticum aestivum) and three our interest species (Asparagus ocinalis, Dendrobium catenatum, Phalaenopsis equestris) belonging to Asparagales order of ower plants and another woody ornamental plant with many existed studies, Rosa chinensis. We used HMMER search method to identify more SBT family members in these species, and additionally performed phylogenetic analysis to uncover the evolutionary relationship of SBT in these eight species. Besides, we also analyze gene structure, chromosomal distribution, orthologous and paralogous, duplication, and expression pattern at the genome level for SBT. This study will lay the foundation for exploring the evolutionary relationship and biological function of SBT family members in plants. Results Identication of SBTs in eight plant species We used HMMER search (based on Peptidase S8 domain) to identied a total of 702 genes in the eight plant species, Arabidopsis thaliana, Oryza sativa, Zea mays, Triticum aestivum, Asparagus ocinalis, Dendrobium catenatum, Phalaenopsis equestris and Rosa chinensis. The detailed information about selected SBT genes is available in Additional le 1. And the identied numbers were 57 in Arabidopsis thaliana, 63 in Oryza sativa, 66 in Zea mays, 255 in Triticum aestivum, 75 in Asparagus ocinalis, 71 in Dendrobium catenatum, 51 in Phalaenopsis equestris and 64 in Rosa chinensis, respectively. Notably, through our used method, the numbers of identied genes in Arabidopsis and rice are similar same to the past two researches, 56 in Arabidopsis (Rautengarten et al 2005) and 63 in rice (Tripathi & Sowdhamini 2006), respectively. Multiple sequence alignment of SBTs in eight plant species Page 3/22 The total of 702 genes and protein sequence were compared and the multiple sequence alignment result was shown as Fig1. All the SBT protein sequences were listed in the Additional le 2. And the similarity comparison matrix was shown in Additional le 3, which was used to draw the alignment heatmap. Fig1, the alignment heatmap, showed that many SBTs shared their sequences with high similarity. However, there were still some sequences showed less similarity with others. This indicated some SBT genes could have specic function in life activity. In addition, we selected the Peptidase S8 domain from Arabidopsis and rice to compare sequence similarity (Fig S1). The result showed that the Peptidase S8 domain displayed high similarity in some sequence areas, but in some other areas, the similarity exposed very low. This signposted conserved feature of Peptidase S8 domain but it was also variable in Arabidopsis and rice. Phylogenetic analysis and classication of SBT genes in eight plant species Additionally, the phylogenetic analysis indicated that SBTs can be divided into 9 clades among the eight plants (Fig 2). The subtrees with gene names are shown in Fig. S2. Clade mainly contains the SBTs from Asparagus ocinalis, Dendrobium catenatum, Phalaenopsis equestris, these three spices belonging to Asparagales. Clade is mainly constructed with subfamilies of SBT in wheat and maize. Clade contains less subfamily members of SBT with some genes in wheat, rice, maize, Asparagus ocinalis, Dendrobium catenatum, Phalaenopsis equestris and Arabidopsis AtSBT 1.2. Clade contains SBT genes from the 7 spices including rice OsSub19 gene and Arabidopsis Acetyl-Coa Carboxylase 2 (ACC2) gene (This gene has a similar protein domain as peptidase S8 domain). Clade is a bigger clade with more SBT genes mainly from monocotyledon, with Arabidopsis SBT6.1 as well. Clade is constructed mainly with Arabidopsis and wheat SBT genes. In this Clade, it has Arabidopsis SBT genes like AtSBT3.5, AtSBT 3.11, AtSBT5.1 and wheat TraesCS7B02G015300. Clade is formed mainly with Arabidopsis “AtSBT4” genes (like AtSBT 4.1 and AtSBT4.9) , wheat and Rosa chinensis SBT genes. Clade contains SBT genes from the 7 plants, and the Arabidopsis SBT gene AtSBT2.5 is noticeable. Clade mainly contains SBT genes from wheat. We compared the SBTs’ sequences from Arabidopsis and rice (Fig S2). The sequences showed the serval conserved domains in the front and the latter part of SBTs. That indicated SBTs function were conserved in some way. Motif and gene structure analysis of SBTs gene family in Arabidopsis and rice Because